High-throughput analysis of sulfatides in cerebrospinal fluid using automated extraction and UPLC-MS/MS

Abstract

This article is available online at http://www.jlr.org ceramide (sphingoid base and a fatty acid) and a sulfated galactose. STs are, together with galactosylceramides, the most typical myelin lipids, where the long-chain fatty acid moieties C24:0 and C24:1 are expressed in particular (1, 2). In addition to myelin, STs have been shown to be expressed in subpopulations of neurons and astrocytes in the gray matter of the brain (3, 4). STs and galactosylceramides are required for the stability and maintenance of the myelin sheet (5), whereas the STs found in other brain regions suggest more diverse functions of these glycosphingolipids (6). Furthermore, STs have been suggested to participate in other cellular processes, such as protein trafficking, cell adhesion, and aggregation and immune responses, among others (7). STs are also present in many other organs, including kidney, liver, heart, intestine, muscle, and pancreas (8), although in minor amounts compared with brain, except in kidney.Findings indicate that the release of STs from myelin is associated with the development of CNS diseases and that STs in cerebrospinal fluid (CSF) might function as a marker of demyelination, myelin damage, and myelin turnover (9-13). Changes in CSF and brain ST levels have also been observed in other neurological disorders, such as early Alzheimer's disease (AD) (14-16). Furthermore, the absence of hydroxylated forms of ST in CSF could be used as a diagnostic tool for fatty acid 2-hydroxylase deficiency (17). These examples accentuate the need for a robust and sensitive method for quantification of STs in CSF.In previous studies, CSF ST measurements have usually been performed by TLC with monoclonal antibody Abstract Sulfatides (STs) are a group of glycosphingolipids that are highly expressed in brain. Due to their importance for normal brain function and their potential involvement in neurological diseases, development of accurate and sensitive methods for their determination is needed. Here we describe a high-throughput oriented and quantitative method for the determination of STs in cerebrospinal fluid (CSF). The STs were extracted using a fully automated liquid/liquid extraction method and quantified using ultra-performance liquid chromatography coupled to tandem mass spectrometry. With the high sensitivity of the developed method, quantification of 20 ST species from only 100 l of CSF was performed. Validation of the method showed that the STs were extracted with high recovery (90%) and could be determined with low inter-and intra-day variation. Our method was applied to a patient cohort of subjects with an Alzheimer's disease biomarker profile. Although the total ST levels were unaltered compared with an age-matched control group, we show that the ratio of hydroxylated/nonhydroxylated STs was increased in the patient cohort. In conclusion, we believe that the fast, sensitive, and accurate method described in this study is a powerful new tool for the determination of STs in clinical as well as preclinical settings.-Blomqvi...

Sulfatide isoform pattern in cerebrospinal fluid discriminates progressive MS from relapsing‐remitting MS

“…We exclude age as a determinant of sulfatide levels in CSF as we have previously shown, employing a cohort of HD with a broader age range, that sulfatide levels in CSF do not increase with age (Blomqvist et al . ).…”

“…As shown before (Blomqvist et al . ), the sulfatide isoforms carried a range of fatty acids, varying in chain length, hydroxylation, and degree of unsaturation. The total sulfatide levels in CSF from PMS patients were significantly higher compared with those determined in CSF from RRMS patients (97.8 ± 9.6 vs. 77.4 ± 3.8 nM; p = 0.044).…”

“…The quantification of sulfatide isoforms was performed as previously described (Blomqvist et al . ). No blinding of the samples was performed, however, the sample preparation and analysis of sulfatides were performed after simple randomization of samples.…”

“…(Blomqvist et al . ). Sulfatide C19:0 isoform was used as an internal standard and quantification was made using a native sulfatide mixture with known total sulfatide content and isoform composition.…”

“…In this study, we have used a recently developed method, based on highly sensitive ultra‐performance liquid chromatography coupled to tandem mass spectrometry (UPLC‐MS/MS) (Blomqvist et al . ), to investigate the concentrations of sulfatide isoforms in CSF from newly diagnosed RRMS and PMS patients and compared them to CSF obtained from healthy donors (HD).…”